Abnormal gene expression in PD

该项目的总体目标是确定在基因中经历异常水平表达的基因 受帕金森病(PD)影响的神经元，并导致其死亡。其基本原理是帕金森病和其他神经退行性变模型中的神经元死亡需要转录依赖的基因调控。一旦这些基因被识别并确定它们在神经元死亡中的因果作用，它们将成为帕金森病预防/改善治疗的潜在靶点。在最后一个时期，我们使用SAGE检测到大约1200个转录本(14,000个转录本中)在细胞培养PD模型中显著上调。在其他发现中，SAGE档案显示PD模拟物诱导内质网应激反应，这可能导致死亡。对于下一阶段，我们提出了以下具体目标：1继续挖掘我们的SAGE数据中的6-OHDA反应基因。生物信息学将用于将额外的SAGE标签与已知转录本进行匹配，并提供对受调控基因在神经元死亡和/或帕金森病中潜在功能作用的最新评估。2.继续SAGE鉴定的精选6-OHDA反应基因的功能研究。基因选择将反映推测的与帕金森病和神经元死亡的潜在相关性。最初的重点将是内质网应激基因，特别是促凋亡转录因子CHOP。已经选择了另外八个受调控的基因作为最初的重点。研究将包括用来自野生型和突变小鼠的PC12细胞和培养的交感神经元进行功能丧失和功能获得的实验。当合适的动物模型(空的或转基因的)可用时，将与我们的合作者Burke和Przedborski博士一起开发这些模型。3.继续检测在我们的SAGE研究中检测到的反应基因在帕金森病动物模型和帕金森病组织中是否也上调。动物模型将与Burke博士和Przedborski博士合作进行检查。PD和对照组织将通过a)检查。 用激光捕获显微切割技术从SNPC和交感神经元(SCG)中提取RNA，用定量聚合酶链式反应(PCR)检测SNPC和交感神经元(SCG)的免疫组织化学。4.使用对照神经元和PD神经元的SAGE或MPSS产生的数据来检测额外的调控转录本，以研究PD神经元死亡的因果作用。这一较长期的目标将寻求利用对照和帕金森病神经元的基因图谱。SCG、SNPC和PC12细胞的图谱比较将为上述体外和体内功能分析的基因鉴定提供一个强大的过滤器。

This project's overall goal is to identify genes that undergo abnormal levels of expression in neurons affected by Parkinson Disease (PD) and that contribute to their death. The rationale is that neuron death in models of PD and other neurodegenerations requires transcription-dependent gene regulation. Once such genes are recognized and their causal roles in neuron death established, they will become potential targets for preventive/ameliorative therapy in PD. During the last period, we used SAGE to detect approximately 1,200 transcripts (out of 14,000) that are significantly up-regulated in a cell culture PD model. Among other findings, the SAGE profile revealed that PD mimetics induce an endoplasmic reticulum stress response that may contribute to death. For the next period, we propose the following specific aims: 1 To continue to "mine" our SAGE data of 6-OHDA responsive genes. Bioinformatics will be used to match additional SAGE tags to known transcripts and to provide updated assessments of the potential functional roles of the regulated genes in neuronal death and/or PD. 2. To continue studies of the functional roles of select 6-OHDA-responsive genes identified by SAGE. Gene selection will reflect deduced potential relevance to PD and neuron death. Initial emphasis will be on ER stress genes and in particular on the pro-apoptotic transcription factor CHOP. Eight additional regulated genes have been chosen for initial emphasis. Studies will include loss- and gain-of-function experiments with PC12 cells and sympathetic neurons cultured from wild-type and mutant mice. When suitable animal models (null or transgenic) become available, these will be exploited with our collaborators Drs. Burke and Przedborski. 3. To continue examining whether responsive genes detected in our SAGE study are also up-regulated in animals models of PD and in PD tissue. Animal models will be examined collaboratively with Drs. Burke and Przedborski. PD and control tissue will be examined by a) immmunohistochemistry of SNpc and sympathetic neurons (SCG) and b) by quantitative PCR using RNA from such neurons harvested by laser capture microdissection. 4. To use data generated by SAGE or MPSS of control and PD neurons to detect additional regulated transcripts for studies of causal roles in neuronal death in PD. This longer-range aim that will seek to exploit gene profiling of control and PD neurons. Comparison of profiles from SCG, SNpc and PC12 cells will provide a powerful filter for identification of genes for functional in vitro and in vivo analyses as described above.